Radiation remains a primary treatment modality for many forms of cancer and can be highly effective. New technologies are allowing more targeted delivery and demonstrating that single or limited fractions of high doses may be more effective than the conventional, multi-fraction, low doses in controlling tumor growth. An important component of this control appears to be the immune system's response to malignant cells and opens up the possibility of combining radio and immunotherapy. A critical part of the immune response to tumors is the capacity of immune cells to infiltrate into the tumor and function within this unique microenvironment, which in many cases has been shown to be immunosuppressive. The goal of this research is to study the balance of immunosuppressive and immunostimulatory factors present within the tumor microenvironment, determine how local radiation therapy alters this balance, and delineate the cellular and molecular mediators involved in this process. The proposed studies are based on our previous work demonstrating that local irradiation of a murine tumor model results in an enhancement of the anti-tumor immune response including alteration in the blood vessels and increased immune cell infiltration and function. We hypothesize that the balance of factors is critical in regulating the overall immune response and determining the efficacy of radiotherapy. Understanding how this balance is altered by local radiation and the cellular and molecular mediators involved will provide the information needed to tilt the balance toward immunostimulation. The first aim will focus on immunostimulatory cytokines including the type 1 interferons and IL-12, and explore how they influence the levels and activity of interferon-gamma (IFN-3) and IFN-3-inducible genes, which we believe are key factors in initiating and sustaining anti-tumor immunity. Particularly interesting is the striking ability to control tumors with a combination treatment of IL-12 and high dose radiation. We will explore the mechanisms of this tumor control, which may involve both direct effects on the tumor cells and the tumor vasculature, such as VCAM-1 upregulation, as well as stimulatory effects on the immune cells. Finally, we will investigate the therapeutic potential of each cytokine in promoting tumor control. The second aim will focus on the role of immunosuppressive factors that are present post radiation therapy. We will study the molecular mediators that may impede the antitumor activity of radiation-induced IFN-3 and explore ways to limit this inhibition. We will determine the radiation sensitivity of T regulatory cells, an important class of T cells that may severely limit effective anti-tumor immunity. The proposed studies will utilize genetically targeted mice to assist in verifying mechanisms, as well as advances we have made in fluorescence microscopy and real time imaging of vessels and immune cells within tumors. The information obtained will provide insight into the interplay between malignant cells and cells of the immune system both before and after radiation therapy, information essential for effective combination of these modalities. PUBLIC HEALTH RELEVANCE: The proposed studies will provide critical information about the induction of immunostimulatory and immunosuppressive factors that result from radiation therapy. This information will allow for therapeutic intervention to shift the response in favor of immunostimulation, thus allowing for the control of malignant cells and limiting tumor growth.